Below pH 3 pepsinogen, which has no other known enzymatic activity, cleaves one of its own peptide bonds near the amino terminus in an intramolecular reaction that generates an active protease. The proposal will investigate this phenomenon, which is a general one for aspartate protease zymogens. Every phase that is experimentally accessible will be studied. Data have already been collected for three of the steps: an early conformational change detected in stopped-flow experiments as a change in fluorescence, formation of the binding site detected in stopped-flow experiments by the environmentally sensitive fluorescence of an active site-directed reagent, and the dissociation of the activation peptide detected by a change in ESR spectrum of a pepsinogen derivative spin labeled near the amino terminus. An additional step will be observed, the peptide bond cleavage, detected by quenching activating mixtures into a denaturant at timed intervals and separation and quantitation of the products to determine the rate of this event. In addition to focusing on these functional events, the proposed work will look at conformational changes of the activation peptide sequence during activation an correlate these changes with the functional steps listed above. For this a fluorescent group will be attached to the zymogen near its amino terminus and fluorescence energy transfer from the bulk of the protein to the activation peptide sequence will be observed in stopped-flow experiments. Evidence is presented to demonstrate that there are two parallel activation pathways. It would appear that pepsinogen during its activation may appear in two general conformations of roughly equal stability. Altering the pH can shift the balance between the two conformations; this makes the relative importance of the two pathways vary in a regular manner with pH. The proposed work will also investigate the effects of temperature and neutral salts drawn from the lyotropic series on the proportionation of activation between the two pathways at each of the functional steps listed above. The result will be a complete description of pesinogen activation in kinetic and in mechanistic terms.